Analyses of blood samples for the presence or absence of antibodies or antigens are used in the diagnosis of diseases, such as HIV infection, hepatitis, Lyme disease, prenatal profiles including TORCH (an acronym for: "Toxoplasmosis, Rubella, Cytomegalovirus, Herpes") profiles, as well as other infectious disease profiles. Presently, such serologic diagnoses are often performed by fluorescent immunoassay. Immunofluorescent assays are not only used for detection of antigens and antibodies in infectious diseases but are widely used for detection of autoimmune phenomena and for localizing pathologically altered serum or tissue elements, e.g., Graves disease, connective tissue diseases, multiple sclerosis, kidney disease, myasthenia gravis, pemphigus, pemphigoid, as well as many pathologic conditions such as cirrhosis of the liver, vasculitis etc. Analyses may also be performed to measure the levels of hormones such as insulin, thyroxine, thyroid stimulating hormone (TSH), blood coagulation factors, factor VIII, yon Willebrand factor; and levels of other possible blood constituents, such as digoxin, morphine, and also blood vitamins such as B.sub.12 and folic acid; as well as any other substances which may be present in small amounts in the biologic sample, which substances have a specific binder available.
Non-immunologic binding pairs include systems wherein the two components share a natural affinity for each other but are not antibodies. Exemplary non-immunologic pairs are biotin/streptavidin or biotin/avidin; intrinsic factor/vitamin B.sub.12 ; folic acid/folate binding protein; hormone/hormone receptor; nucleic acid complexes; antibody/antibody binding protein, i.e., IgG/protein A; DNA/DNA; DNA/RNA/; carbohydrates/lectins; complementary peptide sequences; complementary nucleotide sequences; effector/receptor molecules; enzyme cofactors/enzymes; enzyme inhibitors/enzymes; a peptide sequence/antibodies specific for that sequence protein; polymeric acids/bases; dyes/protein binders; peptides/specific protein binders; and the like.
In a standard indirect fluorescent immunoassay, an antigen, which is the coupling partner for the antibody to be detected, i.e. float tissue culture cells for DNA antigen, or tissue (skin, liver, muscle, etc) or any microorganism or virus, is first affixed to a solid support medium such as a glass slide. With soluble antigens, a paper membrane, nitrocellulose or nylon membrane (Dot blot Technique) can be used. A sample of serum from the patient is then allowed to incubate in contact with the affixed antigen for a period of time sufficient for the partner antibody, if present, to become attached to the affixed antigen. The support surface is then washed to remove all unbound antibodies. A reagent consisting of a labelled antibody to human immune (antibody) globulins is next brought into contact with the support surface and incubated for a time sufficient to cause linkage of the labelled material and any traces of the patient's antibodies which may have bound to the fixed antigen. The excess reagent is then washed off and the support surface is examined to determine if any label is present. Examination of the prepared sample, depending upon the nature of the label, can be done visually; or by spectrophotometry or fluorometry; or by radiation detection means.
It will be appreciated that the aforesaid procedure requires multiple specimen handling steps, including washing, and analysis techniques, and is thus labor intensive and time-consuming. The aforesaid procedure is usually used to detect the presence or absence of only one antigen-specific antibody per test but under certain circumstances two different fluorescent dye labels (fluorescein and rhodamine) are used if one suspects that at least two antibodies may be implicated and characteristic in the suspected disease, i.e., basement membrane antibody as well as DNA antibody, and the substrate used is, for example, a skin section. Usually an antiglobulin is used as the second antibody, however if it is desired to ascertain if the antibody present in the body fluid is IgG or IgM, this can be done using specific anti-IgG or IgM or for that matter any anti-immunoglobulin. Specific immunoglobulins are often used to determine placental transfer of disease, such as syphilis, viral infections, etc, in order to ascertain if the disease is in early or late phases, or to determine whether a patient is producing a specific immunoglobulin to an antigenic stimulus. Fluorescent immunoassays for hormones, drugs and proteins of diagnostic importance have also been developed.